To date the production of the majority of recombinant proteins is performed intracellularly in E.coli. To obtain the product in a usable form one has first to disrupt the cells and then to perform elaborate purification.
The purification can be greatly simplified by using a host/gene combination that leads to secretion of the expressed protein product. E.coli has often been the microorganism of choice. Since not many proteins are known to be naturally secreted by Gram-negative bacteria there is a tendency to start using Gram-positive bacteria such as Bacilli in the production of foreign proteins.
Expression levels of proteins bound to be secreted and originating from Gram-positive bacteria in both homologous and non-homologous Gram-positive hosts have often been satisfactory, yielding about 0.5-1 g/l of protein. However, yields in Gram-positive hosts are substantially lower upon expression of secretory or exported proteins from Gram-negative bacterial or eukaryotic Origin (Palva 1989). Different explanations have been given for this phenomenon and attempts have been made to enhance the yields of foreign proteins.
A primary reason for the low yields was assumed to be the extracellular proteolysis of the expression products. Much effort has therefore been spent on constructing protease-negative hosts (Kawamura and Doi 1984, Fahnestock and Fisher 1987, Sloma et al. 1988), the secretion problems however, partly remained.
Another explanation for the low yields upon expression of foreign proteins may be a rate-limiting step in the intracellular processing. The effect of such a rate-limiting step will be more pronounced if the foreign protein is overexpressed. Generally, either homologous or heterologous genes are introduced into the host cell on high copy number cloning vehicles or integrated into the genome. A strong promoter is cloned upstream of the gene or the gene is integrated downstream of a strong promoter. The introduction of such a construct may give rise to a heavy burden on the translational or secretional apparatus of the cells. Possible rate-limiting steps may be, for example, transcription, translation, intracellular transport, translocation and finally the actual release into the culture medium. Translocation and the actual release into the medium are the subject of the present invention. An important role in membrane transport of proteins is played by specific sequences in the protein.
Many secreted and membrane bound proteins are synthesized in a precursor form. This precursor contains an N-terminal addition of 15-30 amino acids, the signal or leader peptide. There is great variability as to the length and the sequence of these peptides. However, there are some general structural characteristics that must be satisfied in order for these peptides to correctly perform their function. Signal peptides have a basic amino-terminal region followed by a central hydrophobic core that may span the membrane. At the C-terminus there usually is a small uncharged amino acid.
Most of the present knowledge concerning signal peptidases (SPases) in prokaryotic systems has been derived from studies in E.coli. In this organism at least two different SPases can be distinguished. SPase I (synonymous for leader peptidase as used in this text), is capable of processing most of the proteins. A notable exception are glyceride modified lipoproteins (Tokunaga et al. 1982), which are processed by SPase II, also known as prolipoprotein signal peptidase (Tokunaga et al. 1982, Yamada et al. 1984, Yamagata et al. 1982).
The isolation and cloning of the E.coli SPase I (lep) gene is described by Date and Wickner (1981). Aliquots of cell lysates from individual colonies of a complete genomic E.coli DNA library in ColE1 plasmids were assayed for their ability to convert M13 procoat to coat protein posttranslationally. Thus, a strain could be detected that overproduced SPase I. The growth behavior of this strain (7-47) was comparable to that of other strains in the collection. Restriction fragments of pLC7-47 were recloned in pBR322. A 30-fold increase in SPase I concentration was detected in one of the strains after transformation of the plasmids into E.coli. Upon infection of this overproducing E.coli strain with M13 an increase in the transformation of procoat (precursor) to coat (integral transmembrane) protein could be detected. No effect on periplasmatic or secreted proteins was described.
The sequence of the SPase I-encoding gene (lep) from E.coli was determined by Wolfe et al. (1983), who also determined that this protein is largely found in the inner membrane.
Dalbey and Wickner (1985) have cloned and expressed the E.coli lep gene under control of the arabinose promoter and could not detect any effect on protein translocation upon expression of the lep gene. Upon repression of SPase I synthesis they found that cleavage of the signal sequence was essential for the release of the proteins from the membrane.
The effect of overproduction of the SPase I described in the above references was only determined on the M13 integral transmembrane protein. The effect on outer membrane or exported proteins was not described.
The effect of overproduction of cloned SPase I on a periplasmic (TEM beta-lactamase) and an outer membrane protein (PhoE) was reported by Anba et al. (1986). They showed that overproduction did not result in any increase in processing rates for either one of the mentioned proteins and therefore concluded that the SPase I is not the rate-limiting component with the subject precursors and under the conditions that were used.
In all of the above references the effect of SPase I overproduction on homologous proteins with their natural signal sequences was studied. Furthermore, to date the cloning and expression of the lep gene of only one species e.g. E.coli has been described.
In view of the advantages described above with respect to the use of Gram-positive bacteria in the production of recombinant proteins it could be very useful to clone and overexpress signal peptidase genes from other species then E. coli, particularly from Gram-positive bacteria. Although it may be expected that the homology between signal peptidase encoding genes from Gram-positive and Gram-negative bacteria may be sufficient for cross-hybridization Lampen et al. (1986) reported that they could not obtain reproducible signals upon hybridization of the E. coli lep gene with genomic DNA from Bacilli and Staphylococcus aureus in Southern blotting experiments.
As indicated above it can be expected that processing efficiency may become a rate-limiting step in the secretion of overproduced proteins. Palva (1989) suggested that it would be interesting to test whether the cloning of a signal peptidase gene or some other component of the translocation machinery would further increase production yield. However, no suggestion was made on how to perform this.